PROJECT SUMMARY Altered cellular metabolism is a hallmark of cancer as tumors rewire metabolic pathways to meet the bioenergetic, biosynthetic, and redox requirements of malignant cells. Metabolic reprogramming is achieved mainly by mutation of oncogenes and/or tumor suppressors. In non-small cell lung cancer (NSCLC), the most prevalent form of lung cancer, oncogenic mutation of KRAS and loss of function mutation of STK11 (which encodes the tumor suppressor LKB1) is particularly common (6-12%). This combination of mutations specifies both aggressive oncological behavior and metabolic perturbation. It is my central hypothesis that this concurrent mutation alters metabolic preferences in NSCLC, rendering mutant cells dependent on a suite of enzymes and pathways. In support of this hypothesis, I recently discovered that KRAS/LKB1 co-mutant cells share metabolomic signatures of perturbed nitrogen handling. In particular, KRAS/LKB1 co-mutants are addicted to a urea cycle enzyme, carbamoyl phosphate synthetase 1 (CPS1). This enzyme produces carbamoyl phosphate (CP) in the mitochondria from ammonia and bicarbonate, initiating nitrogen disposal. The selective dependence of KRAS/LKB1 co-mutants on CPS1, however, is not due to the canonical activity of the enzyme. Instead, CPS1 enables an unconventional pathway of nitrogen flow from ammonia into pyrimidines. CPS1 loss reduces the pyrimidine/purine ratio, induces DNA replication fork stalling and subsequent DNA damage. With our previous studies as background, in this proposal, I will seek to understand the mechanisms contributing to metabolic transformation in an aggressive subset of NSCLC. To this end, I will pursue the following three specific aims: 1) to elucidate the mechanism and regulation of CPS1 addiction, 2) to investigate the mechanism by which oncogenic KRAS mutation contributes to CPS1 addiction in KRAS/LKB1 co-mutants, and 3) to identify additional novel metabolic liabilities associated with nitrogen metabolism in LKB1/KRAS co-mutants. My primary career goal is to obtain a tenure-track position in an academic setting at a major U.S. university. My long-term goal is to build an independent research career as a cancer biologist with a specific focus on metabolism. To achieve these goals, I hope to develop my intellectual knowledge base as well as increase my technical skill repertoire throughout the duration of the proposed study. This training should be readily attainable in the DeBerardinis laboratory at UT Southwestern Medical Center (UTSW); our lab has expertise in the biochemical analysis and molecular dissection of complex metabolic pathways. In order to promote and assure my progress during the K22 Award period, I have organized an advisory committee consisting of internationally renowned scientists with expertise in different areas relevant to my research and career goals. Collectively, the proposed studies will provide significant insight into mechanisms of metabolic liability in cancer and offer potential strategies for targeting a highly aggressive subtype of cancer. Moreover, this work will provide me with the means to establish myself as an independent investigator.